WILDCAT HYBRID SCORING FOR CONSERVATION BREEDING UNDER THE SCOTTISH WILDCAT CONSERVATION ACTION PLAN Dr Helen Senn, Dr Rob Ogden Wildcat Hybrid Scoring For Conservation Breeding under the Scottish Wildcat Conservation Action Plan Dr. Helen Senn1, Dr. Rob Ogden Subjected to academic review and approved by Scottish Wildcat Conservation Action Plan Steering Group May 2015 Citation: Senn HV and Ogden R, Wildcat Hybrid Scoring For Conservation Breeding under the Scottish Wildcat Conservation Action Plan (2015), Royal Zoological Society of Scotland, May 2015 Cover image: Peter Cairns, northshots.com 1 Communicating author [email protected] 2 About Scottish Wildcat Action The Scottish wildcat is one of Europe's most elusive and endangered mammals. Often referred to as the ‘Tiger of the Highlands’, it is one animal whose image we recognise instantly. Striking, handsome and powerful, it is the very essence of a wild predator living by stealth and strength. We have come to the stage where urgent action is needed to save Scotland's remaining wildcats. We have given ourselves just six years to halt the decline. Scottish Wildcat Action is one of the most ambitious conservation projects ever undertaken in Scotland, with over 20 organisations, community groups and landowners coming together to tackle the decline of Scottish wildcats. The work is a key part of delivering the national Scottish Wildcat Conservation Action Plan, and involves both in situ and ex situ conservation activities, including targeted effort in six priority areas, monitoring and surveying work, and a conservation breeding programme (based at RZSS Highland Wildlife Park in Kingussie). Project partners 3 Executive Summary 1. This document is designed to set out the genetic system for determining hybridisation and how it should be integrated with morphological data, in putative specimens of the wildcat (Felis silvestris) destined to be brought into a conservation breeding programme, overseen by the Royal Zoological Society of Scotland as part of the Scottish Wildcat Conservation Action Plan (SNH 2013). 2. Wildcats hybridise with the domestic cat and produce fertile offspring. 3. In the absence of whole genome sequencing, a sample of genetic markers are capable of estimating the extent of hybridism in an individual with an associated degree of confidence. Wildcat phenotype provides further assessments of wildcat ancestry. 4. The test system devised by RZSS to select the animals for conservation breeding with the greatest available proportion of wildcat ancestry, employs 35 nuclear SNP DNA markers and one mitochondrial marker in combination with pelage assessments. The genetic test is based on one of the more powerful (83 SNP) tests currently available, developed in Switzerland, and has the advantage of generating data that can be compared to datasets for wildcats across Europe. It produces very similar estimates of hybridism to the Swiss test, has a slightly lower degree of confidence associated with them, but is faster and more cost-effective to run. 5. Testing of cat samples collected from across Scotland indicates that there is a complete genetic continuum between wild and domestic cat genetic types in the wild/feral cat population. From the relatively limited sampling to date, the wild-living cat population is a hybrid swarm, i.e. most individuals demonstrate some level of hybridisation. 6. Any method of choosing “wildcats” for a conservation breeding programme needs to decide on a cut-off between wildcat and domestic cat types. 7. We suggest that as a general principle we choose cats in which we have a 95% confidence of them being closer than a first generation backcross to wildcat based on their genetic scores. A first generation backcross to wildcat is a cat where one of its four grandparents is a domestic cat and the remaining three are wildcats. 8. Based on the limited evidence currently available, there does not always appear to be good correspondence between the genetic test and the commonly used phenotypic test (pelage score). This is likely because hybridisation in Scotland has been occurring for a long time and the phenotypic traits are under the control of very few genes that do not match the areas examined in the genetics test. However, this correspondence requires further analysis from a larger sample of individuals with a range of phenotypes and genotypes. 9. We propose that genetic and phenotypic tests are used as separate, independent lines of evidence, when decisions are made about cats. Only where the two lines of evidence corroborate should we chose cats for the conservation breeding programme. 4 Contents About Scottish Wildcat Action ............................................................................................................ 2 Executive Summary ............................................................................................................................. 4 Purpose ............................................................................................................................................... 6 History of wildcat hybrid testing ......................................................................................................... 6 Principle of hybrid testing ................................................................................................................... 7 Current test ......................................................................................................................................... 8 mtDNA marker ................................................................................................................................ 8 Nuclear markers .............................................................................................................................. 8 Background justification to the test.................................................................................................... 9 Theoretical limits to the test ........................................................................................................... 9 Reference data and choice of loci ................................................................................................. 11 The performance of statistical methods (STRUCTURE) and the empirical limits to the test ........ 15 Discussion of limitations of the test .............................................................................................. 19 Decisions for hybrid cut-off criteria .................................................................................................. 25 Nuclear genetic criteria at the 35 Locus test ................................................................................ 25 Power of mtDNA test: ................................................................................................................... 28 Pelage Scores ................................................................................................................................ 30 The test decision ............................................................................................................................... 32 The Principle of the test ................................................................................................................ 32 The details ..................................................................................................................................... 32 Pelage Scoring Criteria .................................................................................................................. 32 Genetic Scoring Criteria ................................................................................................................ 32 Combined pelage and genetic scoring decision matrix: ............................................................... 33 References ........................................................................................................................................ 35 Appendices ........................................................................................................................................ 38 Appendix 1: Allele frequencies at the 35 SNPs in the 82 individual test dataset ......................... 38 Appendix 2: STRUCTURE output at different values of K in the 82 individual test dataset.......... 41 Appendix 3: Test data ................................................................................................................... 41 Appendix 4: Physical validation of the test ................................................................................... 46 Appendix 5: Standard Test protocol at RZSS................................................................................. 47 Appendix 6: Nuclear SNP assay information and reorder numbers ............................................. 49 Appendix 7: SNP assay clustering ................................................................................................. 56 5 Purpose This protocol is designed to set out the genetic system for determining hybridisation in putative specimens of the wildcat (Felis silvestris) destined to be brought into a conservation breeding programme overseen by the Royal Zoological Society of Scotland (RZSS) 2 as part of the Scottish Wildcat Conservation Action Plan (SNH 2013). Wildcats hybridise with the domestic cat (Felis catus) and produce fertile offspring. Genetic tests are capable of determining the extent of hybridism in an individual with a degree of confidence. This document